Detecting potato viruses using direct reverse transcription quantitative PCR (DiRT-qPCR) without RNA purification: an alternative to DAS-ELISA
Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost–benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqMan® qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used.
KeywordsPotato leaf roll virus (PLRV) Potato virus Y (PVY) Potato virus S (PVS) TaqMan® real-time PCR assay Simple sample preparation Direct reverse transcription quantitative PCR (DiRT-qPCR) Conventional reverse transcription quantitative PCR (CoRT-qPCR)
We thank Dr. Petr Dedic, who worked at the Potato Research Institute Havlíčkův Brod (Czech Republic) for providing virus infected in vitro potato plant material for assay validation and assay sensitivity comparison, Johannes Hertrich for technical assistance and the technical assistance team of the Bavarian State Research Center for Agriculture (Freising, Germany) for conducting the DAS-ELISA assay. In addition, we want to thank Thomas Eckl (Bavarian State Research Center for Agriculture, Freising, Germany) for support in statistical calculation and Dr. Miriam Prinz (Bavarian State Research Center, Freising, Germany) for article proof reading.
We also thank the former head of the Associate Professorship of Fruit Science Prof. Dr. Dieter Treutter who passed away and the current temporary head Prof. Dr. Wilfried Schwab for their support.
This work was funded by the Bavarian State Ministry for Food, Agriculture and Forestry (Stmelf). Furthermore, there was a financial contribution by the National Association of seed potato growers Bayern e. V. (Landesverband der Saatkartoffel-Erzeuger-Vereinigungen in Bayern e.V.) and by the potato growers of the German Society for Plant Breeding e.V. (Gesellschaft für Pflanzenzüchtung e.V., GPZ).
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